Proteins are the principal molecular machines of life and have myriad functions including catalysis, transport, sensing, and structure. Loss or impairment of protein function is the ultimate cause of many disease states. Conversely, proteins are the major weapons with which disease-causing organisms attack humans and are a major target of drug research efforts. One limitation to complete understanding of the molecular basis of disease is ignorance concerning the relationship between the amino acid sequence, conformation, stability, and activity of a protein. The link between conformation and stability, in particular, has received considerable attention and is the focus of the proposed research. The long term aim of this research is a quantitative understanding of the relationship between sequence, conformation, and thermal stability in proteins. To this end, stabilizing interactions in ovomucoid domains, a family of small (Mr 6000) protein proteinase inhibitors, are being identified and quantified. Two experimental approaches are used: definition of stable subdomains within turkey ovomucoid third domain (OMTKY3) using peptide hydrogen (NH) exchange and identification of electrostatic interactions through determination of pKa values and the pH dependence of OMTKY3 stability. The proposed research will extend tests of NH exchange that appears to be reporting the denaturation equilibrium and address hypotheses concerning additional conformational equilibria. The specific aims here are: l) measure NH exchange in oligopeptides derived from OMTKY3, 2) measure exchange at solvent-exposed NHs in native OMTKY3, 3) measure the energetics of OMTKY3 stability at alkaline pH using calorimetry, and 4) measure slowed NH exchange from OMTKY3 at alkaline pH. The extraordinary stability of OMTKY3 to alkaline pH raises the possibility of using NH exchange in the native protein to measure the kinetics of conformational changes, including protein folding, on a microsecond time-scale. The molecular origin of the perturbed carboxyl pKas will be investigated through l) determination of lysine pK as in OMTKY3 and 2) mutagenic substitution of lysines thought to interact with these carboxyl groups. These studies will also permit further tests of the relationship between perturbed pKas and the pH dependence of OMTKY3 stability.